Precise temporal and spatial control over cell shape changes and cell rearrangements are essential for the development of all metazoans. Endocrine signaling provides a critical level of temporal control that additionally aids in the coordination of morphogenetic events. The elongation and eversion of Drosophila leg imaginal discs provide an ideal system for defining the molecular mechanisms of hormone-regulated tissue morphogenesis. A pulse of the steroid hormone ecdysone triggers the transformation of an epithelial sac into an immature adult leg through coordinated changes in cell shape. Previous studies indicate that ecdysone-dependent leg morphogenesis requires signaling through the Rho 1 small GTPase. Although the direct effects that Rho 1 signaling has on the actin cytoskeleton are well established, little is known about the mechanisms that control Rho 1 activity in a developmental context. Understanding these control mechanisms for Rho 1 signaling are critical since perturbation of Rho activity during vertebrate embryonic development can lead to incomplete neural tube closure and heart defects. Furthermore, dysregulation of Rho signaling at later stages of development may result in tumor progression and metastasis. The overall goal of the proposed research is to define the morphogenetic pathway of leg development in Drosophila, from an initiating signal that provides temporal specificity, through a Rho 1 intracellular signaling cascade, to the molecules that apply chemomechanical forces to the actin cytoskeleton. This will be accomplished through the development of methods to monitor Rho 1 activation in imaginal discs undergoing morphogenesis. These methods will be used to examine the activation of Rho 1 by ecdysone. In addition, whole Drosophila genome microarray chips will be probed to identify genes induced by ecdysone at the onset of leg morphogenesis, and simultaneous genetic screens will be conducted to identify Rho 1-interacting genes. It is expected that these genetic and molecular screens will converge to identify key genes that regulate Rho 1 signaling and leg morphogenesis, providing a foundation for understanding how Rho 1 is regulated during a developmental program in an intact living organism, as well as providing insights into how Rho signaling can be subverted to a pathological state. [unreadable] [unreadable] [unreadable]